Thyrotropin, or thyroid stimulating hormone (TSH), is a pituitary hormone that regulates thyroid function via the TSHR (Szkudlinski M W, Fremont V, Ronin C, Weintraub B D 2002 Thyroid-stimulating hormone and TSHR structure-function relationships. Physiological Reviews 82: 473-502). The TSHR is a G-protein coupled receptor and is composed of three domains:—a leucine rich domain (LRD), a cleavage domain (CD) and a transmembrane domain (TMD) (Nunez Miguel R, Sanders J, Jeffreys J, Depraetere H, Evans M, Richards T, Blundell T L, Rees Smith B, Furmaniak J 2004 Analysis of the thyrotropin receptor-thyrotropin interaction by comparative modelling. Thyroid 14: 991-1011). Binding of TSH to the TSHR triggers receptor signalling which leads to stimulation of formation and release of thyroid hormones; thyroxine (T4) and tri-iodothyronine (T3). A negative feedback mechanism involving the levels of T4 and T3 in the circulation controls the release of TSH from the pituitary (and thyrotropin releasing hormone secreted by the hypothalamus) that in turn controls thyroid stimulation and the levels of thyroid hormones in serum.
It is well documented in the art that some patients with autoimmune thyroid disease (AITD) have autoantibodies reactive with the TSHR (Rees Smith B, McLachlan S M, Furmaniak J 1988 Autoantibodies to the thyrotropin receptor. Endocrine Reviews 9: 106-121). In a majority of cases, these autoantibodies bind to the TSHR and mimic the actions of TSH thereby stimulating the thyroid to produce high levels of T4 and T3. These autoantibodies are described as thyroid stimulating autoantibodies or TSHR autoantibodies (TRAbs) with stimulating activity or TSH agonist activity. The physiological feedback mechanism of thyroid function control mentioned above is not effective in the presence of such thyroid stimulating autoantibodies and patients present with symptoms of thyroid hyperactivity or thyrotoxicosis (excess of thyroid hormones in serum). This condition is known as Graves' disease. In some patients, TRAbs with stimulating activity are thought to be responsible for interaction with TSHRs in retro-orbital tissues and to contribute to the eye signs of Graves' disease. A human monoclonal autoantibody which acts as a powerful thyroid stimulator (hMAb TSHR1) has been described in detail in patent application WO2004/050708A2.
In contrast in some patients with AITD, autoantibodies bind to the TSHR, prevent TSH from binding to the receptor but do not have the ability to stimulate the TSHR. These types of autoantibody are known as TRAbs with blocking activity or TSH antagonist activity, and patients who have blocking TRAbs in their serum may present with symptoms of an under-active thyroid (hypothyroidism) (Rees Smith B, McLachlan S M, Furmaniak J 1988 Autoantibodies to the thyrotropin receptor. Endocrine Reviews 9: 106-121). In particular, TRAbs with blocking activity when present in serum of pregnant women cross the placenta and may block foetal thyroid TSHRs leading to neonatal hypothyroidism and serious consequences for development. Furthermore, TRAbs with blocking activity can be found in breast milk of affected mothers and this may contribute further to clinical hypothyroidism in the baby. To date human monoclonal autoantibodies to the TSHR with TSH antagonist activity have not been available. Consequently, detailed studies of how this type of autoantibody interacts with the TSHR, and how their interactions with the TSHR compare with those of stimulating type of autoantibodies (such as M22) and with TSH, have been limited.
Human chorionic gonadotropin is a hormone produced during pregnancy which has mild thyroid stimulating effects.
Characterisation of the properties of TRAbs with stimulating or blocking activities is of critical importance in studies which aim to improve the diagnosis and management of diseases associated with an autoimmune response to the TSHR. The invention described in patent application WO2004/050708A2 provides details about the properties of a human monoclonal autoantibody with powerful stimulating activity and its interaction with the TSHR. Furthermore, patent application WO2006/016121A discloses a mutated TSHR preparation including at least one point mutation which can be used in the differential screening and identification of patient serum stimulating TSHR autoantibodies, patient serum blocking TSHR autoantibodies and TSH in a sample of body fluid from a patient being screened. Patent application WO2004/050708A2 also describes a mouse monoclonal antibody (9D33) with TSHR blocking activity. 9D33 binds to the TSHR with high affinity (2×1010 L/mol) and is an effective antagonist of TSH, hMAb TSHR1 (M22) and patient serum TRAbs with stimulating or blocking activities (patent application WO2004/050708A2 and Sanders J, Allen F, Jeffreys J, Bolton J, Richards T, Depraetere H, Nakatake N, Evans M, Kiddie A, Premawardhana L D, Chirgadze D Y, Miguel R N, Blundell T L, Furmaniak J, Rees Smith B 2005 Characteristics of a monoclonal antibody to the thyrotropin receptor that acts as a powerful thyroid-stimulating autoantibody antagonist. Thyroid 15: 672-682). Although the mouse monoclonal antibody 9D33 shows at least some of the characteristics of patient serum TRAbs with blocking activity, it is a mouse antibody generated by immunisation of an experimental animal with the TSHR and as such may not be truly representative of TSHR autoantibodies generated in the process of an autoimmune response to the TSHR in humans. As a mouse monoclonal antibody, 9D33 would need to be humanised for in vivo applications in humans. This may be disadvantageous in view of the expense and complication involved in the humanisation process.
The present invention results from the production and properties of a human monoclonal autoantibody (5C9) to the TSHR that is an effective antagonist of TSH and of stimulating TRAbs in patient sera. 5C9 has been isolated from the peripheral lymphocytes of a patient with hypothyroidism and high levels of TSHR autoantibodies. The lymphocytes were immortalised by infection with Epstein Barr virus (EBV) and positive clones fused with a mouse/human cell line to generate a stable clone. IgG was purified from supernatants of clone cultures and the ability of 5C9 IgG to bind to the TSHR and influence TSHR activity was assessed. In particular, the ability of 5C9 to inhibit TSH binding to the TSHR, and to inhibit cyclic AMP stimulating activity of TSH was studied. Furthermore, the ability of 5C9 to inhibit binding of stimulating or blocking patient serum TRAbs to the TSHR and to inhibit their biological activity was also assessed. In addition, the use of 5C9 in assays for TSHR antibodies, TSH and related compounds was investigated. Variable region (V region) genes of the heavy (HC) and light chains (LC) of 5C9 were sequenced and the complementarity determining regions (CDRs) assigned.